**4. Discussion**

As a vector-transmitted parasite, *Leishmania* must adapt to vastly different environments, carbon sources, and antimicrobial defense mechanisms. This must be achieved without differentially regulated RNA synthesis [3,44,45]. Instead, *Leishmania* relies on modulated RNA stability [46], RNA processing [47] and inducible translation [2,5,48] as means of short-term gene expression control. Long-term adaption to changing environments, by contrast appears to be mediated by gene copy number variations, either by chromosomal aneuploidy [6,7] or by amplification of genes and gene clusters [36,49,50]. A third level of gene expression control are PTMs of proteins that may activate or inhibit activities or influence localization. Examples of PTMs are protein kinase mediated phosphorylation of threonine and serine side chains [8,11,13]. Side chain-specific modifications can impact on protein folding or protein–protein interactions. The covalent attachment of modifying polypeptides is another, as yet

little understood mode of expression control in *Leishmania*. So far, only the impact of a ubiquitin fold modifier (UFM1) protein was demonstrated [17,51] in *L. donovani*. A similar modifier, small ubiquitin-like modifier (SUMO) was identified and characterized in *Trypanosoma* spp. where it is involved in surface antigen expression and nuclear organization [18,19,52]. Here we describe the *Leishmania* SUMO and SENP orthologs and characterize them by reverse genetic, biochemical and cell biological means.

To the best of our knowledge, SUMO is an essential gene in *L. donovani* promastigotes. Attempts to produce SUMO−/<sup>−</sup> null mutants by CRISPR-mediated gene editing failed while the same gene replacement tools were successfully employed in a strain carrying ectopic SUMO copies (Figure 2D,E), indicating that null mutants are either non-viable or non-proliferative as promastigotes *in vitro*. It was shown for higher eukaryotes that the SUMO pathways are essential during differentiation processes [53,54], but our literature search did not turn up reports of an outright SUMO gene replacement. This is probably also due to the presence of multiple SUMO genes in mammalian cells [55], which may confound reverse genetics approaches.

Unlike SUMO, SENP appears to be non-essential, albeit with a significant impact on promastigote proliferation at optimal growth temperature, with a smaller effect at mammalian tissue temperatures. Fittingly, the survival of amastigotes within mouse macrophages is unaffected by the loss of SENP. This may indicate an important role of SENP and its clients during logarithmic growth of *Leishmania* promastigotes, but less impact during the slow growth of intracellular amastigotes. Yet, with SUMO C-terminal processing abrogated by the loss of SENP (Figure 4C) and its nuclear localization severely reduced (Figure 5), it surprises that the effect of SENP loss is not equally deleterious as the loss of SUMO. Strong signals for C-terminally tagged SUMO in SENP null mutants (Figures 4C and 5) argue against a SUMO processing pathway using alternative proteases. One must therefore assume, that apart from its role as a conjugated protein modifier, SUMO must have additional, essential functions in *Leishmania*.

SUMO and its processing protease, SENP, are proteins with constitutive, above-average synthesis rates in *Leishmania*, indicating a need for abundance or a high turnover rate. Indeed, SUMO (LinJ.08.0480) showed little changes of abundance during promastigote-to-amastigote differentiation *in vitro* [21], and SENP (LinJ.26.2070) has a constitutive abundance too [24].

Immune fluorescence microscopy of tagged SUMO protein shows a nuclear, but not kinetoplast localization. This localization fully depends on SENP-mediated C-terminal processing (Figure 5). This result is in keeping with reports that show involvement of SUMO in nuclear organization and chromosome segregation [53]. Preliminary data (A.B.), however, show no impact of a SENP loss on the accessibility of *L. donovani* chromatin to micrococcal nuclease digest. This must be seen, however, in the context of the Trypanosomatida having a divergent chromatin structure and nuclear architecture. While the genomic DNA is assembled into 10 nm fibers of nucleosomes, these protozoa lack further condensation of chromosomes into 30 nm solenoid fibers [56]. The function of SUMO in the nucleus may therefore be diverged.

The affinity of HA-tagged SUMO for the nucleus is also a promising possibility to identify SUMOylated proteins from the cytoplasm and the nucleus via immune precipitation of SUMO-target conjugates and subsequent mass spectrometric analysis.
